Leukotriene-A4 hydrolase

 

Leukotriene A4 hydrolase/ Aminopeptidase is involved in the immune response in humans, and is unique in its ability to catalyse two very different reactions at the active site. Not only is it able to catalyse the hydrolysis of the epoxide Leukotriene A4 to produce the potent immune activator Leukotriene B4, it is also able to catalyse the hydrolysis of peptide bonds, acting on the first peptide bond after the N terminal. One active site is enough to confer such variety of function, so the enzyme represents a yardstick of evolutionary efficiency seldom reached in nature. It displays a characteristic Zinc binding motif at the active site (GXMEN) which places it in the family M1 of Zinc metalloproteases.

 

Reference Protein and Structure

Sequence
P09960 UniProt (3.3.2.6, 3.4.11.4) IPR012777 (Sequence Homologues) (PDB Homologues)
Biological species
Homo sapiens (Human) Uniprot
PDB
1hs6 - STRUCTURE OF LEUKOTRIENE A4 HYDROLASE COMPLEXED WITH BESTATIN. (1.95 Å) PDBe PDBsum 1hs6
Catalytic CATH Domains
1.10.390.10 CATHdb 1.10.1740.60 CATHdb (see all for 1hs6)
Cofactors
Zinc(2+) (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:3.3.2.6)

leukotriene A4(1-)
CHEBI:57463ChEBI
+
water
CHEBI:15377ChEBI
→
leukotriene B4(1-)
CHEBI:57461ChEBI
Alternative enzyme names: LTA4H, LTA(4) hydrolase, Leukotriene-A4 hydrolase, LTA-4 hydrolase, Leukotriene A(4) hydrolase,

Enzyme Mechanism

Introduction

In the hydrolysis of Leukotriene, a water molecule, activated by Zinc acts as an acid to protonate the epoxide, causing ring opening. This leads to a carbocation, stabilised by delocalisation between the three double bonds. Nucleophilic attack by the OH- ion, activated this time by Glu 271, on the terminal double bond of the delocalised system leads to the final product Leukotriene B4. In the hydrolysis of peptides, a water molecule, activated by Glu 296 and Zinc, attacks the peptide bond leading to a tetrahedral intermediate with negative charge localised to the oxygen. This is stabilised by binding to Zn2+ and by favourable contacts between the N terminal section and Glu 271. Protonation of the leaving group by Tyr 383 leads to collapse of the intermediate and the formation of the products.

Catalytic Residues Roles

UniProt PDB* (1hs6)
Glu297 Glu296(297)A Activates a water molecule for nucleophilic attack by deprotonation in the peptidase reaction. In this mechanism it activates and stabilises the reactive intermediates formed. electrostatic stabiliser
Glu272 Glu271(272)A Has a vital role in both reaction. In the hydrolysis of the epoxide, acts to activate a water molecule towards nucleophilic attack. In the hydrolysis of the aminopeptide, acts to stabilise the tetrahedral intermediate by contact with the amino terminal. hydrogen bond acceptor, electrostatic stabiliser
Tyr384 Tyr383(384)A Primarily functions as a general acid/base in the peptidase reaction by protonating the leaving group thus allowing collapse of the tetrahedral intermediate to occur. In this mechanism it likely helps to stabilise the intermediates formed. electrostatic stabiliser
Asp376 Asp375(376)A Acts as a general acid/base during the course of the reaction. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
His300, His296, Glu319 His299(300)A, His295(296)A, Glu318(319)A Forms part of the zinc binding site. metal ligand
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

proton transfer, overall reactant used, intermediate formation, heterolysis, decyclisation, charge delocalisation, bimolecular nucleophilic addition, intermediate terminated, overall product formed, native state of enzyme regenerated, inferred reaction step

References

  1. Rudberg PC et al. (2002), J Biol Chem, 277, 1398-1404. Leukotriene A4Hydrolase/Aminopeptidase: GLUTAMATE 271 IS A CATALYTIC RESIDUE WITH SPECIFIC ROLES IN TWO DISTINCT ENZYME MECHANISMS. DOI:10.1074/jbc.m106577200. PMID:11675384.
  2. Haeggström JZ et al. (2007), Prostaglandins Other Lipid Mediat, 83, 198-202. Structure and catalytic mechanisms of leukotriene A4 hydrolase. DOI:10.1016/j.prostaglandins.2007.01.006. PMID:17481555.
  3. Rudberg PC et al. (2002), Proc Natl Acad Sci U S A, 99, 4215-4220. Leukotriene A4 hydrolase: Selective abrogation of leukotriene B4 formation by mutation of aspartic acid 375. DOI:10.1073/pnas.072090099. PMID:11917124.
  4. Thunnissen MM et al. (2001), Nat Struct Biol, 8, 131-135. Crystal structure of human leukotriene A(4) hydrolase, a bifunctional enzyme in inflammation. DOI:10.1038/84117. PMID:11175901.

Step 1. The oxygen of the oxirane ring in leukotriene A4 deprotonates a water molecule activated by Glu271.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp375(376)A hydrogen bond acceptor
Glu271(272)A hydrogen bond acceptor, electrostatic stabiliser
His299(300)A metal ligand
His295(296)A metal ligand
Glu318(319)A metal ligand

Chemical Components

proton transfer, overall reactant used, intermediate formation

Step 2. The acidic oxirane ring collapses in a heterolysis.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp375(376)A hydrogen bond acceptor
Glu271(272)A hydrogen bond acceptor, electrostatic stabiliser
Glu296(297)A electrostatic stabiliser
Tyr383(384)A electrostatic stabiliser
His299(300)A metal ligand
His295(296)A metal ligand
Glu318(319)A metal ligand

Chemical Components

heterolysis, decyclisation, intermediate formation, charge delocalisation

Step 3. Asp375 deprotonates a water molecule, which attacks the intermediate in a nucleophilic addition with attendant double bond rearrangement, producing the leukotriene B4 product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu296(297)A electrostatic stabiliser
Tyr383(384)A electrostatic stabiliser
His299(300)A metal ligand
His295(296)A metal ligand
Glu318(319)A metal ligand
Asp375(376)A hydrogen bond acceptor
Glu271(272)A hydrogen bond acceptor, electrostatic stabiliser
Asp375(376)A proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, overall reactant used, intermediate terminated, overall product formed

Step 4. Water deprotonates Asp375, regenerating the enzyme active site.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His299(300)A metal ligand
His295(296)A metal ligand
Glu318(319)A metal ligand
Asp375(376)A hydrogen bond donor, proton donor

Chemical Components

proton transfer, native state of enzyme regenerated, inferred reaction step
Download: Image, Marvin File

Step 1. The oxygen of the oxirane ring in leukotriene A4 deprotonates a water molecule activated by Glu271.

Step 2. The acidic oxirane ring collapses in a heterolysis.

Step 3. Asp375 deprotonates a water molecule, which attacks the intermediate in a nucleophilic addition with attendant double bond rearrangement, producing the leukotriene B4 product.

Step 4. Water deprotonates Asp375, regenerating the enzyme active site.

Products.

Introduction

This proposal proceeds via an ester intermediate. In this scheme, the zinc alone activates and opens the epoxide, and the carboxylate of Glu-271 attacks LTA4 at C6 to form an ester intermediate. In a concerted SN2′ reaction, this ester can then be attacked by an hydroxyl group or a carboxylate at C12, and the negative charge can move along the conjugated triene system, eventually leading to an alkyl-oxygen cleavage instead of a normal ester cleavage.

Catalytic Residues Roles

UniProt PDB* (1hs6)
Glu297 Glu296(297)A Activates the water molecule that donates its proton to the reactive intermediate. It also activates and stabilises the reactive intermediates formed. electrostatic stabiliser
Glu272 Glu271(272)A Acts as a nucleophile and forms a covalent bond with the substrate. hydrogen bond acceptor, nucleofuge, nucleophile
Tyr384 Tyr383(384)A In this mechanism it likely helps to stabilise the intermediates formed. electrostatic stabiliser
Asp376 Asp375(376)A Acts as a general acid/base during the course of the reaction. hydrogen bond acceptor, proton acceptor
His300, His296, Glu319 His299(300)A, His295(296)A, Glu318(319)A Forms part of the zinc binding site. metal ligand
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

proton transfer, overall reactant used, intermediate formation, intermediate terminated, overall product formed, bimolecular nucleophilic substitution with allylic rearrangement, native state of enzyme regenerated

References

  1. Rudberg PC et al. (2002), J Biol Chem, 277, 1398-1404. Leukotriene A4Hydrolase/Aminopeptidase: GLUTAMATE 271 IS A CATALYTIC RESIDUE WITH SPECIFIC ROLES IN TWO DISTINCT ENZYME MECHANISMS. DOI:10.1074/jbc.m106577200. PMID:11675384.

Step 1. Glu271 initiates a nucleophilic attack on the epoxide group.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp375(376)A hydrogen bond acceptor
His299(300)A metal ligand
His295(296)A metal ligand
Glu318(319)A metal ligand
Glu271(272)A nucleophile

Chemical Components

proton transfer, overall reactant used, intermediate formation

Step 2. Asp375 deprotonates a water molecule, which attacks the intermediate in a nucleophilic addition with attendant double bond rearrangement, producing the leukotriene B4 product with concomitant deprotonation of a zinc bound water.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr383(384)A electrostatic stabiliser
Glu296(297)A electrostatic stabiliser
Glu271(272)A hydrogen bond acceptor
His299(300)A metal ligand
His295(296)A metal ligand
Glu318(319)A metal ligand
Asp375(376)A proton acceptor
Glu271(272)A nucleofuge

Chemical Components

proton transfer, overall reactant used, intermediate terminated, overall product formed, ingold: bimolecular nucleophilic substitution with allylic rearrangement, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Glu271 initiates a nucleophilic attack on the epoxide group.

Step 2. Asp375 deprotonates a water molecule, which attacks the intermediate in a nucleophilic addition with attendant double bond rearrangement, producing the leukotriene B4 product with concomitant deprotonation of a zinc bound water.

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid, Peter Sarkies